CN109182456A - Method based on hybridization chain reaction and dynamic light scattering detection urine Telomerase Activity - Google Patents
Method based on hybridization chain reaction and dynamic light scattering detection urine Telomerase Activity Download PDFInfo
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- CN109182456A CN109182456A CN201811086677.6A CN201811086677A CN109182456A CN 109182456 A CN109182456 A CN 109182456A CN 201811086677 A CN201811086677 A CN 201811086677A CN 109182456 A CN109182456 A CN 109182456A
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
- C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
Abstract
The method based on hybridization chain reaction (HCR) and dynamic light scattering (DLS) detection urine Telomerase Activity that the invention discloses a kind of.This method is in the presence of active Telomerase, and the extension product of substrate can cause probe H1 and H2 and HCR reaction occurs, and forms long, notched double stranded DNA product.Then HCR product can hybridize with gold nano grain (DNA-AuNP) probe of DNA modification, and gold nano grain is caused to be assembled.The variation of gold nano grain dynamic light scattering signal (particle size) is measured, realizes the highly sensitive quantitative analysis to telomerase activation.This method is easy to operate, testing cost is low, required sample is few, high sensitivity and specificity it is good.Meanwhile this method is used for the detection of kinds cancer patient urine Telomerase Activity by we, the results showed that, the present invention has specificly-response to bladder cancer, provides new approach for the non-invasive diagnosis of bladder cancer.
Description
Technical field
The present invention relates to a kind of activity test method of telomerase, belong to cancer non-invasive diagnostic techniques field, and in particular to one
Method of the kind based on hybridization chain reaction and dynamic light scattering detection urine Telomerase Activity.
Background technique
Bladder cancer is a kind of common malignant disease, is the second common tumour in urogenital system, it has also become male
In the most common malignant tumour fourth, the 9th in women.The diagnostic method of bladder cancer mainly includes cystoscopy and urine
Cytolgical examination.Cystoscopy is to diagnose the standard method of urinary system symptom, but its higher cost, and patient can be allowed to generate
Discomfort causes complication.Urinary cytology inspection is a kind of method of Noninvasive, its specificity with higher, but by
Cast-off cells are less in urine, especially during rudimentary and infantile tumour, cause its sensitivity lower.Therefore, a kind of
Noninvasive, simple, reliable Diagnosis of Bladder method is highly desirable.
In recent years, some Noninvasives carried out for urine, which detect, to be studied, such as telomeric repeat amplification (TRAP) method
Detect level of telomerase activity, detected by comet assay Urine exfoliative cell DNA damage, urine DNA methylation analysis etc..However, these
Biomarker needs further progress research to verify.
Telomerase is a kind of ribonucleoprotein complex, it can pass through its own intrinsic RNA template and reverse transcriptase
TTAGGG repetitive sequence is added in telomerase with GAP-associated protein GAP and maintains telomere length.Telomerase activation is in most of normal humans
It is suppressed in tissue, telomere length successively decreases with the period division of cell, leads to the aging and death of cell.But 85% or more
Malignant cell Telomerase Activity be activated and express, be proliferated cancer cell constantly.Therefore, Telomerase is considered
It is a kind of potential Tumor biomarkers, is a kind of very promising therapy target.
The main method of telomerase activation detection at present is the telomeric repeatamplification protocol (Telomeric of based on PCR
Repeat Amplification Protocol, TRAP).However, the method, there are complicated for operation, technique is cumbersome, somewhat expensive with
And the problems such as non-specific amplification.
Therefore, the detection for how developing a kind of simple, sensitive method to realize telomerase activation be it is necessary,
It is one of ordinary skill in the art's technical problem urgently to be solved.
Summary of the invention
Regarding to the issue above and demand, the present invention provides a kind of based on HCR and DLS detection urine Telomerase Activity
Method realizes the Gao Ling to telomerase activation by measuring the variation of gold nano grain dynamic light scattering signal (particle size)
Quick quantitative analysis.
In order to solve the above-mentioned technical problem, the technical solution adopted by the present invention is as follows.
A method of urine Telomerase Activity being detected based on hybridization chain reaction and dynamic light scattering, main includes such as
Lower step:
1) gold nano grain (DNA-AuNP) probe of DNA modification is prepared；
2) extension: be added in containing Telomerase primer to be measured (TS primer), dNTPs and amplification buffer to
Telomere zyme extract is surveyed, constant-temperature incubation makes TS primer that extension occur, obtains extension reaction mixture；
3) HCR reacts: two hairpin probe H1 and H2 being added in extension reaction mixture, it is anti-to carry out HCR after mixing
It answers, obtains long, notched double stranded DNA product；
4) DNA-AuNP probe is added in HCR product, assembles gold nano grain by DNA hybridization.
The method of above-mentioned detection urine Telomerase Activity, in step 2), under the catalysis of sample to be tested telomerase, TS
The extension products of primer can induce H1 and H2 that HCR reaction occurs, and the TS primer not extended cannot cause HCR reaction.
The method of above-mentioned detection urine Telomerase Activity, the TS primer have nucleotide shown in SEQ ID NO:1
Sequence (5`-AAT CCG TCG AGC AGA GTT-3`), the sequence of extension products are 5`-AAT CCG TCG AGC AGA
GTT AGGG TTA GGG-3`。
The method of above-mentioned detection urine Telomerase Activity, in step 3), hairpin probe H1 and H2 include two parts, and one
Divide and participate in HCR reaction, another part can hybridize with DNA-AuNP probe.
The method of above-mentioned detection urine Telomerase Activity, the hairpin probe H1 have nucleosides shown in SEQ ID NO:2
Acid sequence (5`-CCC TAA CCC TAA CTC TGC TCGA AAG AGA TCG AGC AGA GTT AGGG TT TTT
TTT TTT TTT-3`)；
Probe H2 has nucleotide sequence (5`-TCG AGC AGA GTT AGGG TTA GGG shown in SEQ ID NO:3
CCC TAA CTC TGC TCGA TCT CTT TT TTT TTT TTT TTT-3`)。
The method of above-mentioned detection urine Telomerase Activity, in step 4), the DNA sequence dna of gold nano particle modification is according to hair
The complementary fragment of folder probe H1 and H2 are designed；According to the dynamic light scattering signal (particle size) of gold nano grain, measurement
Telomerase activation in sample.
The method of above-mentioned detection urine Telomerase Activity, DNA-AuNP probe have nucleotide shown in SEQ ID NO:4
Sequence (HS-5`-AAA AAA AAA AAA AAA AAA AA).
The method of above-mentioned detection urine Telomerase Activity, further includes step 5), measures gold nano grain dynamic light scattering
The highly sensitive quantitative analysis to telomerase activation is realized in the variation of signal (particle size).
The method of above-mentioned detection urine Telomerase Activity, the Monitoring lower-cut of this method are 4 cells；This method is especially suitable
Urine detection for bladder cancer patients.
The method of above-mentioned detection urine Telomerase Activity, the extraction process of urine telomerase are as follows:
Fresh urine sample is collected at 4 DEG C, is centrifuged 10 minutes under 850rpm, after being rinsed once with PBS again at 2300rpm, 4 DEG C
Precipitating, is then dissolved in 200 μ l lysis buffers by centrifugation 5 minutes, and is incubated for 30 minutes on ice, finally at 4 DEG C,
Be centrifuged 20 minutes under 12000rpm, supernatant shifted, be stored in -80 DEG C it is spare.
By above-mentioned technical proposal, the invention has the advantages that and advantageous effects:
1) method of the invention is in the presence of active Telomerase, and the extension product of primer (TS Primer) can cause
HCR reaction, HCR product can hybridize with DNA-AuNP probe, and gold nano grain is caused to be assembled, by measuring gold nano
The highly sensitive quantitative analysis to telomerase activation is realized in the variation of grain dynamic light scattering signal (particle size).
2) method of the invention has many advantages, such as easy to operate, high sensitivity, at low cost, and this method is particularly suitable for
The urine detection of bladder cancer patients.
3) the result shows that, the present invention to bladder cancer have specificly-response, for bladder cancer non-invasive diagnosis provide it is new
Approach.
Detailed description of the invention
Attached drawing is used to a further understanding of the present invention, and constitutes part of specification, with the embodiment of the present invention one
It rises and is used to explain the present invention, be not construed as limiting the invention.
Fig. 1 is that the present invention is based on the method schematics that HCR and dynamic light scattering detect urine Telomerase Activity.
The present invention is shown in Fig. 2 a and Fig. 2 b respectively (is not caused HCR and drawn using dynamic scattering analysis difference sample
Send out HCR) particle size distribution figure.
Fig. 3 is the particle size figure of present invention detection different number MCF-7 Cell Telomerase Activity, and illustration is 10 to 1000
The standard curve of a MCF-7 cell.
Fig. 4 is the particle size figure of present invention detection various cancers patient urine telomerase activation.
Specific embodiment
The invention discloses one kind based on telomere in hybridization chain reaction (HCR) and dynamic light scattering (DLS) detection urine
The method of enzymatic activity.
Hybridization chain reaction (hybridization chain reaction, HCR) is put as a kind of isothermal, without enzymatic nucleic acid
Big technology is concerned, and is to make two hairpin probes that cascade hybridization reaction occur under the induction for causing chain, forms long, band notch
Duplex DNA.HCR can be in conjunction with a variety of probes, and gold nano grain (gold nanoparticles, AuNPs) is a kind of
It is flat to be widely used in sensing due to its large specific surface area, optical property uniqueness, good biocompatibility for most common nano material
The building of platform.AuNPs also changes therewith with the change of dispersion/coherent condition, particle size, therefore, can by AuNPs with
HCR is combined, and gold nano grain particle size can be converted to the amount of HCR product.
Dynamic light scattering (Dynamic Light Scattering, DLS) is a kind of common optical measuring technique, and measurement is straight
Particle size of the diameter between 0.5nm~6 μm, the size range of most of nano materials is in the ideal detection range of DLS, gold
The particle size of nano particle can be detected using DLS.
As shown in Figure 1, for the present invention is based on the method schematics that HCR and DLS detects urine Telomerase Activity.When not having
When Telomerase or Telomerase inactivate, gold nano grain keeps dispersity；And in the presence of active Telomerase, gold nano grain hair
Raw aggregation, partial size significantly increase.
This method is in the presence of active Telomerase, and the extension product of substrate can cause probe H1 and H2It is anti-that HCR occurs
It answers, forms long, notched double stranded DNA product.Then HCR product can be with the gold nano grain (DNA- of DNA modification
AuNP) probe hybridizes, and gold nano grain is caused to be assembled.Measure gold nano grain dynamic light scattering signal (particle size)
The highly sensitive quantitative analysis to telomerase activation is realized in variation.
Method of the invention is easy to operate, testing cost is low, required sample is few, high sensitivity and specificity it is good.Meanwhile it should
The Monitoring lower-cut of method is 4 cells；This method is used for the detection of kinds cancer patient urine Telomerase Activity by we.
Below by way of specific preferred embodiment combination attached drawing, invention is further described in detail, but the present invention and not only
It is limited to embodiment below.
As shown in Figure 1, the present invention relates to a kind of sides for detecting urine Telomerase Activity based on HCR and dynamic light scattering
Method.It is specific as follows:
1, the property of TTAGGG repetitive sequence can be added in the Telomerase primer end (TS primer) based on Telomerase:
The extension products of TS primer can cause HCR reaction, and TS primer has nucleotides sequence shown in SEQ ID NO:1
It arranges (5`-AAT CCG TCG AGC AGA GTT-3`), the sequence of extension products is 5`-AAT CCG TCG AGCAGA GTT
AGGG(TTA GGG)n-3`；
2, HCR reacts:
Extension products and hairpin probe H1 (had into nucleotide sequence 5`- shown in SEQ ID NO:2CCC TAA CCC TAA CTC TGC TCGA AAG AGA TCG AGC AGA GTT AGGG TT TTT TTT TTT TTT-3`) and H2 (have
Nucleotide sequence 5`- shown in SEQ ID NO:3TCG AGC AGA GTT AGGG TTA GGG CCC TAA CTC TGC TCGA TCT CTTTT TTT TTT TTT TTT-3`) mixing progress HCR reaction (dashed part is to participate in HCR reactive moieties), it obtains
Long, notched HCR product；
3, hybridize:
Preparing DNA-AuNP probe (has nucleotide sequence HS-5`-AAA AAA AAA AAA shown in SEQ ID NO:4
AAA AAA AA), DNA-AuNP probe is added in HCR product, assembles gold nano grain by DNA hybridization；
4, dynamic scattering analysis: the variation of measurement gold nano grain dynamic light scattering signal (particle size), realization pair
The analysis of telomerase activation and the diagnosis of bladder cancer.
As shown in figures 2 a and 2b, respectively it is shown that the present invention (does not cause HCR using dynamic scattering analysis difference sample
With the particle size distribution figure of initiation HCR).
When not having Telomerase or Telomerase inactivates, HCR reaction cannot be caused, the partial size of gold nano grain is 56.4nm；
And in the presence of active Telomerase, the extension products of primer can cause HCR reaction, and the partial size of gold nano grain increases to
347.5nm。
As shown in figure 3, showing the particle size of present invention detection different number MCF-7 Cell Telomerase Activity in Fig. 3
Figure, illustration are the standard curve of 10 to 1000 MCF-7 cells.
The cell number of MCF-7 and the partial size of gold nano grain are in good linear relationship, inspection within the scope of 10 to 1000
Rising limit is 4 cells.As shown in figure 4, showing the partial size of present invention detection various cancers patient urine telomerase activation in Fig. 4
Size figure.
The partial size of only bladder cancer's urine sample significantly increases, and the urine sample of normal person and other cancer patients
Partial size no significant change compared with the partial size of blank sample (Granzyme), show the present invention to bladder cancer have specificity
Response.
Embodiment 1
A method of urine Telomerase Activity is detected based on hybridization chain reaction (HCR) and dynamic light scattering (DLS),
Specifically comprise the following steps:
1) extraction of Telomerase
The extraction of cell telomerase: MCF-7 cell is being contained into 10% fetal calf serum, 100 μ g/ml penicillin and 100 μ
It is cultivated in the DMEM culture medium of g/ml streptomysin.After extracting cell with trypsase, 7 × 10 are had collected6A cell.By cell point
It is dispersed in 1.5 milliliters of EP pipes, 5min is centrifuged at 1000rpm, and washed twice with ice-cold PBS.Extracting solution is suspended in again
200 μ L lysis buffers are centrifuged 20 minutes at 4 DEG C, 12000rpm after being incubated for 30 minutes on ice.Supernatant is shifted, it is dilute
It releases, is stored in spare at -80 DEG C.
The extraction of urine telomerase: collecting fresh urine sample at 4 DEG C, be centrifuged 10 minutes under 850rpm, is rinsed with PBS primary
It is centrifuged 5 minutes, then precipitating is dissolved in 200 μ l lysis buffers, and be incubated for 30 on ice at 2300rpm, 4 DEG C again afterwards
Minute, finally at 4 DEG C, be centrifuged 20 minutes under 12000rpm, supernatant shifted, be stored in -80 DEG C it is spare.
2) the gold nano grain preparation of DNA modification
The preparation of AuNPs is and to have done adjustment appropriate according to classical reduction of sodium citrate method.By 2mL Fresh
38.8mM sodium citrate solution be added rapidly to the 1mM gold chloride HAuCl that 20mL boils4In solution, reaction solution is by yellowish discoloration
For black, then become purple, eventually becomes claret, continue to be heated to reflux and stir 10 minutes.Finally, reaction solution is allowed to be cooled to
Room temperature is simultaneously kept stirring, and is then filtered, is stored in spare in 4 DEG C of refrigerators with 0.45 micron of nylon leaching film.
AuNPs surface modification DNA: mercapto-modified DNA first uses tricresyl phosphate (2- chloroethyl) ester TCEP to handle, and then presses
The molar ratio of 200:1 is added in previously prepared AuNPs solution, is incubated at room temperature 16 hours.Then next 44
NaCl is added in hour several times and carries out salt aging, the ultimate density of NaCl is 0.1M.Finally, by reaction solution with 13800rpm's
Revolving speed is centrifuged 30 minutes to remove free DNA (being repeated 3 times), and obtained oily sediment is dissolved in 10mM PBS buffer solution
In (pH 7.4,0.1M NaCl), it is stored in spare in 4 DEG C of refrigerators.
3) progress of HCR reaction
Telomere zyme extract, 1.5 μM of TS primer, 1mM dNTPs, 0.2mg/mL BSA, 0.4U/mL will be contained
The solution of RNase inhibitor is incubated for 3h at 37 DEG C, and then heating 5 minutes at 95 DEG C makes to inactivate.H1 and H2 is heated at 95 DEG C
5min is cooled to room temperature.Then the solution containing H1 (1 μM), H2 (1 μM) and telomerase extended products was reacted at 37 DEG C
Night.
4) HCR product is mixed with DNA-AuNP probe, and detects change of size.
Appropriate DNA-AuNP probe is added into HCR product, is incubated for 4h at 30 DEG C, Jenner is made by DNA hybridization reaction
Rice grain is assembled, and measures corresponding particle size variation (as shown in figure 3, partial size maximum can increase using dynamic light scattering
It is added to 400nm or so).
The above described is only a preferred embodiment of the present invention, be not intended to limit the present invention in any form, therefore
Without departing from the technical solutions of the present invention, to the above embodiments according to the technical essence of the invention any simply to repair
Change, equivalent variations and modification, all of which are still within the scope of the technical scheme of the invention.
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Claims (10)
1. a kind of method based on hybridization chain reaction and dynamic light scattering detection urine Telomerase Activity, it is characterised in that:
Mainly include the following steps:
1) gold nano grain (DNA-AuNP) probe of DNA modification is prepared；
2) end to be measured extension: is added in containing Telomerase primer to be measured (TS primer), dNTPs and amplification buffer
Granzyme extracting solution, constant-temperature incubation make TS primer that extension occur, obtain extension reaction mixture；
3) HCR reacts: two hairpin probe H1 and H2 being added in extension reaction mixture, carries out HCR reaction after mixing, obtains
To long, notched double stranded DNA product；
4) DNA-AuNP probe is added in HCR product, assembles gold nano grain by DNA hybridization.
2. the method for detection urine Telomerase Activity according to claim 1, it is characterised in that；In step 2), to
Under the catalysis of sample telomerase, the extension products of TS primer can induce H1 and H2 that HCR reaction occurs, and not extend
TS primer cannot cause HCR reaction.
3. the method for detection urine Telomerase Activity according to claim 2, it is characterised in that: the TS primer
With nucleotide sequence shown in SEQ ID NO:1 (5`-AAT CCG TCG AGC AGA GTT-3`), the sequence of extension products is
5`-AAT CCG TCG AGC AGA GTT AGGG (TTA GGG) n-3` (n=1~10).
4. the method for detection urine Telomerase Activity according to claim 1, it is characterised in that: in step 3), hair clip
Probe H1 and H2 include two parts, and a part participates in HCR reaction, and another part can hybridize with DNA-AuNP probe.
5. the method for detection urine Telomerase Activity according to claim 4, it is characterised in that: the hairpin probe H1
With (the 5`-CCC TAA CCC TAA CTC TGC TCGA AAG AGA TCG AGC of nucleotide sequence shown in SEQ ID NO:2
AGA GTT AGGG(T) n-3`) (n=8~24)；
Probe H2 has nucleotide sequence (5`-TCG AGC AGA GTT AGGG TTA GGG CCC shown in SEQ ID NO:3
TAA CTC TGC TCGA TCT CTT (T) n-3`) (n=8~24).
6. the method for detection urine Telomerase Activity according to claim 1, it is characterised in that: in step 4), Jenner
The DNA sequence dna of rice grain modification is designed according to the complementary fragment of hairpin probe H1 and H2；According to the dynamic of gold nano grain
Light scattering signal (particle size) measures the telomerase activation in sample.
7. the method for detection urine Telomerase Activity according to claim 1, it is characterised in that: DNA-AuNP probe tool
There are nucleotide sequence shown in SEQ ID NO:4 (HS-5`- (A) n-3 ') (n=10~30).
8. the method for detection urine Telomerase Activity according to claim 1, which is characterized in that it further include step 5),
The variation of gold nano grain dynamic light scattering signal (particle size) is measured, realizes highly sensitive quantitative point to telomerase activation
Analysis.
9. the method for detection urine Telomerase Activity according to claim 1 to 8, it is characterised in that:
The Monitoring lower-cut of this method is 4 cells；
This method is particularly suitable for the urine detection of bladder cancer patients.
10. the method for detection urine Telomerase Activity according to claim 9, it is characterised in that: urine telomerase
Extraction process it is as follows:
Fresh urine sample is collected at 4 DEG C, is centrifuged 10 minutes under 850rpm, is centrifuged at 2300rpm, 4 DEG C again after being rinsed once with PBS
5 minutes, then precipitating is dissolved in 200 μ l lysis buffers, and is incubated on ice 30 minutes, finally at 4 DEG C, 12000rpm
It is lower centrifugation 20 minutes, supernatant is shifted, be stored in -80 DEG C it is spare.
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| CN109797200A (en) * | 2019-02-13 | 2019-05-24 | 中国科学院苏州生物医学工程技术研究所 | Ratio-type telomere enzyme active quantitive detection method |
| CN109837326A (en) * | 2019-01-17 | 2019-06-04 | 嘉兴学院 | The biological target molecular detecting method of output signal is captured and amplified based on multivalence |
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| CN111693415A (en) * | 2020-06-23 | 2020-09-22 | 广东药科大学 | Telomerase activity detection method based on catalysis hairpin assembly-dynamic light scattering technology |
| CN112795565A (en) * | 2021-02-04 | 2021-05-14 | 南京邮电大学 | Detection probe, kit and direct detection method of telomerase activity |
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| CN113789373A (en) * | 2021-08-24 | 2021-12-14 | 中山大学 | Exosome-based light-controlled signal amplification technology and application thereof in microRNA detection and imaging |
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